DNA methylation assay for colorectal carcinoma
==============================================

* Ji-Jun Chen
* Ai-Qin Wang
* Qing-Qi Chen

## Abstract

Colorectal carcinoma (CRC) is a common cause of morbidity and mortality worldwide. Two pathogenic pathways are involved in the development of adenoma to CRC. The first pathway involves*APC/β-catenin* characterized by chromosomal instability resulting in the accumulation of mutations. The second pathway is characterized by lesions in*DNA mismatch repair genes*. Aberrant DNA methylation in selected gene promoters has emerged as a new epigenetic pathway in CRC development. CRC screening is the most efficient strategy to reduce death. Specific DNA methylation events occur in multistep carcinogenesis. Epigenetic gene silencing is a causative factor of CRC development. DNA methylations have been extensively examined in stool from CRC and precursor lesions. Many methylated genes have been described in CRC and adenoma, although no definite DNA methylation biomarkers panel has been established. Multiple DNA methylation biomarkers, including secreted frizzled-related protein 2, secreted frizzled-related protein 1, tissue factor pathway inhibitor 2, vimentin, and methylguanine DNA methyltransferase, have been further investigated, and observations have revealed that DNA methylation biomarkers exhibit with high sensitivity and specificity. These markers may also be used to diagnose CRC and adenoma in early stages. Real time polymerase chain reaction (qPCR) is sensitive, scalable, specific, reliable, time saving, and cost effective. Stool exfoliated markers provide advantages, including sensitivity and specificity. A stool qPCR methylation test may also be an enhanced tool for CRC and adenoma screening. 

*   Biomarker
*   colorectal carcinoma
*   DNA methylation
*   real time PCR
*   stool

## Introduction

Colorectal carcinoma (CRC) is a common cause of morbidity and mortality worldwide. As a major life-threatening malignancy, CRC ranks second to lung carcinoma in men, third to breast cancer and lung cancer in women, and overall second to other cancer types in men and women. Approximately 142,820 new cases were diagnosed and 50,830 individuals died because of CRC in 2013. Clinical and pathological stages at the time of diagnosis largely determine the prognosis of diagnosed patients. The curative rates of CRC in T1NM, T2NM, and T3NM stages are greater than 90%, 85%, and 70%, respectively. Therefore, CRC screening is an efficient strategy to reduce death caused by this disease. Current screening modalities have resulted in a modest decrease in mortality and failed to achieve high public participation. Among these modalities, highly sensitive colonoscopy is the standard technique used to detect and remove early lesions, but colonoscopy is invasive and costly. For population-wide screening, simple and noninvasive procedures, such as guaiac or immunochemical stool occult blood testing (FOBT), are preferred. However, the performances of these tests are low. As such, molecular tests should be conducted to improve their efficiency. For example, stool aberrant DNA methylation has been developed as a biomarker for CRC initiation and progression. In this article, this technique was discussed to develop real time polymerase chain reaction (qPCR) DNA methylation biomarker test for stool to diagnose CRC. Stool qPCR DNA methylation biomarkers were also examined to predict outcomes and responses to CRC chemotherapy. 

## CRC pathogenesis

Approximately 98% of CRC are adenocarcinomas that almost originate from adenomas as generally curable by resection. The peak incidence age of CRC is 60 to 70 years, and less than 20% of cases occur before the age of 50 years. Genetic and epidemiologic studies have linked CRC to several factors, including inherited predisposition, somatic mutation, dietary influence, and preexisting inflammatory disease1. The development of carcinoma from adenoma is referred to as adenoma-carcinoma sequence. Two major distinct pathogenic pathways are involved in CRC development, and stepwise accumulation of multiple mutations is implicated in these pathways1,2. The first pathway comprises*APC/β-catenin* characterized by chromosomal instability (CIN) that results in the stepwise accumulation of mutations in a series of oncogenes and tumor suppressor genes. The molecular evolution along this pathway occurs through a series of morphologically identifiable stage. The loss of*APC* tumor suppressor gene is the earliest event in adenoma formation. Both copies of the*APC* gene must be lost to stimulate adenoma development. Normal*APC* promotes β-catenin degradation. With loss of*APC*, the accumulated β-catenin translocates to the nucleus and activates the transcription of several genes, such as*MYC* and cyclin D1.*K-RAS* mutations subsequently occur. Further mutation of a putative cancer suppressor gene on 18q21 and*TP53* leads to the final emergence of carcinoma, and additional mutation ensues. The accumulation of mutations rather than their occurrence in a specific order is essential for colorectal carcinogenesis. This adenoma-carcinoma sequence accounts for approximate 80% of sporadic CRC2. The second pathway is characterized by genetic lesions in DNA mismatch repair genes, which are involved in 10% to 15% of sporadic cases. Mutations accumulate, but correlated identifiable morphological characteristics have yet to be determined. Among these DNA mismatch repair genes,*MLH1* is the most commonly involved in carcinogenesis. The loss of*DNA mismatch repair genes* leads to a hypermutable state. In this state, simple repetitive DNA sequences called microsatellites are unstable during DNA replication. This phenomenon causes widespread alterations in these repeats. The resulting microsatellites instability (MSI) is the molecular signature of defective DNA mismatch repair. The loss of mismatch repair then leads to the accumulation of mutations in growth-regulating genes, and thus triggers the emergence of CRC. In addition to these pathways, CpG island methylator phenotype (CIMP) pathway is also involved in CRC development. 

In the majority of patients diagnosed with CRC, carcinoma cells are no longer confined to the primary sites. Approximately 36% exhibit a locally advanced disease and 19% manifest a metastatic disease. The 5-year survival rate of patients with metastatic diseases is 10.3%. CRC incidence and mortality rates have decreased over the past two decades. These trends are consistent with the effectiveness of CRC screening in detecting and removing adenomatous polyps. While these results are encouraging, CRC screening is underused. In general, screening tests can be classified into two categories: invasive (structural) exams and non-invasive tests. Invasive exams can be subdivided into endoscopic techniques (colonoscopy, flexible sigmoidoscopy, and capsule endoscopy) and radiological exams (barium enema, computer tomography colonography and magnetic resonance colonography). Noninvasive tests can be subdivided into tests that detect blood (FOBT) and tests that detect stool DNA3,4. Among these techniques, colonoscopy remains the gold standard, but this technique is invasive, costly, and burdensome. Colonoscopy may also be less protective in the right colon than in the left colon5. FOBT is a simple, non-invasive, relatively cheap, and frequently used screening test. However, FOBT is not designed for precursor lesions detection. Adenomas and CRCs usually cause intermittent bleeding. As such, repetitive testing is required. Guaiac FOBT is weakly sensitive, whereas immunochemical FOBT is highly sensitive. The detection of DNA markers in stool specimens is a relatively new noninvasive screening approach. Multi-targeted assays on 21 specific mutations in the*K-RAS*,*TP53*, and*APC*, which is a MSI marker (*BAT-26*), and DNA integrity assay markers has also been developed. Nevertheless, their overall sensitivities in detecting CRCs and adenomas remain suboptimal6,7, possibly because the accumulation of mutations is essential for colorectal carcinogenesis. The detection of DNA mutation may also fail to reflect the progress of carcinogenesis. 

## DNA methylation and CRC

Epigenetics refers to heritable alterations in gene activity and expression unlikely caused by changes in DNA sequences and potentially reversible self-propagating molecular signatures8. Epigenetics has rapidly expanded as a biological field of study9. The mechanisms of epigenetics include DNA methylation, histone modifications, nucleosome positioning, or noncoding RNA10. Epigenetic modifications, particularly DNA methylation in selected gene promoters, are recognized as common molecular alternations in human tumors. Among epigenetic markers, DNA methylation is the most widely investigated. In DNA methylation, methyl groups are added to the 5-position of cytosine by DNA methyltransferases (DNMT) to produce 5-methylcytosine, which typically represses gene transcription and modifies DNA functions11. Cytosine and adenine in DNA can be methylated. DNA methylation elicits species-specific and tissue-specific effects. For instance, the DNA methylation of cytosine residues in CpG dinucleotides, which are often clustered in so-called CpG islands, leads to transcriptional silencing of the associated genes. Approximately 60% of all human promoters are associated with CpG islands. In the genome of untransformed cells, approximately 90% of all promoters are unmethylated12. The DNA methylation of many genes and their significance in CRC have also been described13. The aberrant methylation of CpG islands within gene promoters and first exonic or intronic regions may induce the transcriptional repression of tumor-suppressor genes. Genes are hypomethylated and hypermethylated in CRC14. Hypermethylation is a discrete targeted event within tumor cells and thus causes specific loss of gene expression. Hypomethylation usually occurs in advanced stages of tumor development and affects genome to a greater extent than hypermethylation does. The most extensively characterized epigenetic alteration is promoter hypermethylation (Figure 1). 

![1](http://www.cancerbiomed.org/https://www.cancerbiomed.org/content/cbm/14/1/42/F1.medium.gif)

[1](http://www.cancerbiomed.org/content/14/1/42/F1)

1 
Mechanisms of DNA methylation in the colorectal carcinoma pathogenesis: the roles of multiple DNA methylation biomarkers in colorectal carcinogenesis.

CIMP is the phenotype of methylated tumor suppressor genes, and tumorigenesis theoretically occurs through progressive genetic silencing even in the absence of genetic mutations15. CIMP is also referred to as an epigenetic phenomenon because this occurrence involves a temporary genetic change in the target DNA sequence, that is, CIMP simply triggers a potentially reversible alteration because of methylation. Approximately 30%–40% of proximal CRCs and 3%–12% of distal CRCs were characterized as CIMP16. CIMP is regarded as a distinct CRC subgroup, which is fundamentally different from other colon cancers. CIMP tumors exhibit unique pathological features, such as high mutations (KRAS or BRAF) rates, proximal location, wild-type p53, mucinous histological type, poor differentiation, and increased occurrence in female and elderly patients17. Primary CRCs can be divided into three subclasses depending on epigenetic and clinical profiles: CIMP1, CIMP2 and CIMP negative. The prognosis of CIMP1 is better than that of CIMP2. CRCs with CIN are likely hypomethylated, whereas CRCs with MSI are hypermethylated. CRCs with neither CIN nor MSI possess a unique methylation pattern and clinical features, including improved prognosis. CIMP mechanisms in the pathogenesis of CRC have yet to be fully elucidated. Hypermethylation secondary to CIMP possibly leads to MSI through MLH1 promoter methylation and subsequent MLH1 mismatch repair gene silencing. DNA methylation has been used as a diagnostic CRC marker because specific methylation events occurring early in multistep carcinogenesis have been identified and epigenetic gene silencing plays a causative role in CRC development. DNA methylation analysis may also provide useful prognostic markers of disease progression and response to traditional chemotherapy. 

The majority of CRCs are adenocarcinomas, which almost originate from adenomas. Adenoma is a neoplastic polyp that ranges from small pedunculated tumors to large sessile lesions. The incidence of adenoma is focused on the colon. All adenomatous lesions arise as the result of epithelial proliferation and dysplasia. Adenomatous polyps are classified into three subtypes on the basis of epithelial architecture: (1) tubular adenomas; (2) villous adenomas; (3) tubulovillous adenomas. The risk of CRC increases when polyps are larger than 2 cm, villous than tubular, and sessile rather than pedunculated. Pedunculated lesions are treated through colonoscopic removal with snare electrocautery. Sessile lesions may require surgical excision. Although colorectal adenomatous polyps are recognized as precursor lesions in most CRC cases, only 1%–10% of individuals with resected adenomas likely develop invasive cancer18. Thus, early detection of colorectal adenoma may help prevent CRC. Adenoma is currently diagnosed with colonoscopy and air contrast barium enema. Aberrant DNA methylation occurs in the stool of adenoma patients. Thus, the feasibility of using DNA methylation biomarkers to diagnose colorectal adenoma should also be evaluated. 

## Multiple DNA methylation biomarkers

DNA methylation biomarkers in stools with CRC and precursor lesions have been extensively examined. Different panels have been reported to improve diagnostic accuracy, although no definite biomarker panel has been established. In our comprehensive analysis of available studies on DNA methylation biomarkers in stools for CRC and adenoma detection, some DNA methylation biomarkers, including secreted frizzled-related protein 2 (SFRP2), secreted frizzled-related protein 1 (SFRP1), tissue factor pathway inhibitor 2 (TFPI2), vimentin, and methylguanine DNA methyltransferase (MGMT), have been investigated. Observations have confirmed that DNA methylation biomarkers are highly sensitive and specific. These markers may also be used to diagnose CRC and adenoma in early stages (Table 1). 

View this table:
[1](http://www.cancerbiomed.org/content/14/1/42/T1)

1 
Major stool DNA methylation biomarkers for colorectal cancer

SFRPs are a family of secreted proteins that can bind to*Wnt* ligands and frizzled receptors and thus modulate the*Wnt* signaling cascades.*Wnt* signaling cascades play an important role in colorectal carcinogenesis and progression19. SFRPs are initially and independently identified as soluble factors implicated in early embryonic development and modulators of apoptotic events. Alterations in SFRP expression levels have been associated with tumor formation and bone and myocardial disorders20. SFRP1 and SFRP2 hypermethylation likely occurs at the onset of all tumor types, including colon carcinomas21–24. SFRP1 hypermethylation may reduce gene expression and contribute to CRC formation25. Epigenetic SFRP1 inactivation is linked to the upregulation of*Wnt*/*β-catenin cascade* in CRC;*Wnt*/*β-catenin* repression has also been considered a mechanism that inhibits tumor cell growth and prevents metastatic invasion (Figure 1)22. SFPR2 and SFRP1 methylation in stool also exnibits high sensitivity and specificity for CRC detection26–40. SFRP2 methylation for CRC identification in stool samples reaches a sensitivity of 90% and specificity of 77%31. A systematic meta-analysis has revealed that the pooled sensitivity and specificity of methylated SFRP2 are 0.71 and 0.94, respectively41. SFRP2 methylation is therefore a promising biomarker for CRC screen42. The DNA stool test of SFRP1hypermethylation also achieves a sensitivity of 89% and specificity of 86% in colorectal neoplasia detection35. 

TFPI2, a member of the Kunitz-type serine proteinase inhibitor family, inhibits the tissue factor/factor VIIa complex and various serine proteinases. The aberrant methylation of TFPI2 promoter CpG islands in human cancer is responsible for the decreased TFPI2 expression during cancer progression. TFPI2 also maintains the stability of tumor environment and inhibits neoplasm invasiveness and growth and metastasis formation (Figure 1)43. TFPI2 methylation in stools also demonstrates high sensitivity and specificity among CRC patients44–46. TFPI2 gene promoter methylation is detected in the stool of CRC patients with a sensitivity of 86.7% and a specificity of 100%46. The sensitivity and specificity of fecal TFPI2 methylation assay for CRC detection range from 76% to 89% and from 79% to 93%, respectively44. 

Vimentin, a major constituent of the intermediate filament family of proteins, is ubiquitously expressed in normal mesenchymal cells and known to maintain cellular integrity. Vimentin has been considered as a marker for epithelial-mesenchymal transition (EMT), although the molecular aspects of vimentin in the function of tumorigenesis remain unknown. The aberrant promoter methylation of the vimentin gene may contribute to colorectal carcinogenesis (Figure 1)47. The promoter hypermethylation of vimentin in stool is also a sensitive, specific alternative for CRC screening38,45,48–52. Aberrant vimentin methylation is detected in fecal DNA from CRC patients, with a sensitivity of 46% and a specificity of 90%51. 

MGMT is a suicide enzyme that repairs the pre-mutagenic, pre-carcinogenic and pre-toxic DNA damage O6-methylguanine. MGMT also repairs large adducts, which are formed in response to pollutants, carcinogens, methylating agent, and chloroethylating anticancer drugs, on the O6-position of guanine. Therefore MGMT is a key node in the defense against commonly found carcinogens and marker of resistance of normal and cancer cells exposed to alkylating therapeutics (Figure 1)53,54. A meta-analysis suggested that the frequency of MGMT hypermethylation is significantly higher than that in CRC, and MGMT gene promoter methylation involved a stepwise carcinogenesis of CRC development55. The loss of MGMT expression, which is secondary to MGMT gene promoter methylation, may increase the responses to alkylating agents56. The methylation stool testing of MGMT is a promising, sensitive, and specific method for early CRC detection30,49,57. Stool-methylated MGMT is detected in 48.1% of CRC patients and 28.6% of adenoma patients30. 

Other stool methylation biomarkers, such as oncostatin M receptor-β, human Mut L homolog-1, hyperplastic polyposis protein gene, SFRP5, GATA4, β-1, 4-galactosyltransferase-1, N-myc downstream-regulated gene 4, 2qI4.2, hypermethylated in cancer 1, ESR1, phosphatase and actin regulator 3, spastic paraplegia-20 and RASSF2, have been proposed to diagnose CRC.

## Perspectives of DNA methylation assay for CRC

Definite DNA methylation biomarker panels haven't yet to be established. Unlike DNA mutation assay, DNA methylation can be measured quantitatively through qPCR, which may be used to monitor disease progression. Purified stool DNA can be chemically modified by sodium bisulfate to convert all unmethylated cytosine to uracils while leaving methylcytosines unaltered. Treated DNA retains methylated cytosines, and shows specific changes in the DNA sequence. Colonocyte shedding from colorectal neoplasm differs quantitatively and qualitatively from that of normal epithelium. Cellular efflux toward the lumen is much greater from CRC than from normal epithelium. Although CRC typically occupies less than 1% of the intestinal surface, tumor-derived DNA in stool from patients with CRC may account for as much as 14% or 24% of the total recovered DNA58,59. Exfoliated coloncytes and colonocyte constituents provide a diverse class of candidate stool markers. Unlike occult bleeding, which occurs as intermittent leaking into the lumen, exfoliated markers are sensitive because of their continuous release. Furthermore, exfoliated markers are potentially specific because they originate from neoplasm per se, where occult blood emanates from circulation. Since the earliest study of abnormal methylation in CRC was reported by Feinberg and Vogelstein60, hundreds of methylated genes have been described in CRC and adenoma. The correct genes to be analyzed should be accurately selected to improve the sensitivity and specificity of DNA methylation tests. 

DNA methylation may provide useful prognostic markers of disease progression and response to therapy. Specific methylation events have been associated with different tumor stages and poor prognoses61. As the first chemotherapeutic agent, 5-fluorouracil (5-FU) has been combined with folinic acid and has been used for CRC chemotherapy. Hence, 5-FU-based therapies remain the standard regimens against advanced CRC. 5-FU inhibits thymidylate synthase, which is a key enzyme in pyrimidines synthesis. Thus, pyrimidines available for DNA replication are reduced. Newly available 5-FU-based oral formulations, such as capecitabine, offer maximum antitumor properties. CIMP status can be assessed as a predictive marker for 5-FU responsiveness62. Cancer-specific DNA methylation events may also be involved in different stages of colorectal carcinogenesis. Unlike gene mutations, DNA methylation may also be reversible. DNA methylation can be affected by single demethylating drug. Chemotherapeutic effect may be predictable prior to drug administration through DNA methylation analysis. Aberrant genes methylation may be essential for the chemosensitivity of CRC to various drugs. The response and chemosensitivity of patients to chemotherapy may also be predicted by determining the status of DNA methylation biomarkers in the stool of CRC patients. 

Recent advancements in engineered DNA-binding molecules, such as transcription activator-like effector (TAL or TALE) proteins, clustered regularly interspaced short palindromic repeats (CRISPR), and CRISPR-associated proteins (Cas) system, have been applied in genome editing in cells63,64. These engineered DNA-binding molecules can bind to a specific DNA sequence and be applied to other purposes. Engineered DNA-binding molecule-mediated chromatin immunoprecipitation has been developed by utilizing a TAL or CRISPR/Cas system to target specific genomic regions and to investigate associated DNA-binding interactions65,66. 

## Conclusions

The development of a sensitive stool qPCR DNA methylation assay for CRC and adenoma can provide a non-invasive, scalable, specific, safe, convenient, and widespread accessible screening tool, which is more acceptable to patients than current commonly used screening methods. This assay may help decrease the morbidity and mortality of CRC. Genomic markers for survival in patients with CRC have yet to be developed. DNA methylation status may be a prognostic factor in patients treated with standard chemotherapy. Patients with high methylation levels may be recommended with alternative treatments.

## Footnotes

*   **Conflict of interest statement** No potential conflicts of interest are disclosed.

*   Received September 29, 2016.
*   Accepted November 7, 2016.


*   Copyright: © 2017, Cancer Biology & Medicine

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This is an open access article distributed under the terms of the [Creative Commons Attribution License (CC BY) 4.0](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.

## References

1.  1. Myers AR.  Medicine. Baltimore: Williams & Wilkins; 1997.
    
    

2.  2. Kumar V, Abbas AK, Aster JC, Robbins SL.  Robbins Basic Pathology. 9th edition. Philadelphia, PA: Elsevier/Saunders; 2013.
    
    

3.  3. de Wijkerslooth TR, Bossuyt PM, Dekker E.  Strategies in screening for colon carcinoma. Neth J Med. 2011; 69: 112–9.
    
    [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=21444935&link_type=MED&atom=%2Fcbm%2F14%2F1%2F42.atom) 

4.  4. Cummings LC, Cooper GS.  Colorectal cancer screening: Update for 2011. Semin Oncol. 2011; 38: 483–9.
    
    [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=21810507&link_type=MED&atom=%2Fcbm%2F14%2F1%2F42.atom) 

5.  5. Brenner H, Hoffmeister M, Arndt V, Stegmaier C, Altenhofen L, Haug U.  Protection from right- and left-sided colorectal neoplasms after colonoscopy: Population-based study. J Natl Cancer Inst. 2010; 102: 89–95.
    
    [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1093/jnci/djp436&link_type=DOI) 
    
    [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=20042716&link_type=MED&atom=%2Fcbm%2F14%2F1%2F42.atom) 
    
    [Web of Science](http://www.cancerbiomed.org/lookup/external-ref?access_num=000274091200008&link_type=ISI) 

6.  6. Imperiale TF, Ransohoff DF, Itzkowitz SH, Turnbull BA, Ross ME, Colorectal Cancer Study Group.  Fecal DNA versus fecal occult blood for colorectal-cancer screening in an average-risk population. N Engl J Med. 2004; 351: 2704–14.
    
    [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1056/NEJMoa033403&link_type=DOI) 
    
    [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=15616205&link_type=MED&atom=%2Fcbm%2F14%2F1%2F42.atom) 
    
    [Web of Science](http://www.cancerbiomed.org/lookup/external-ref?access_num=000225877300006&link_type=ISI) 

7.  7. Ahlquist DA, Sargent DJ, Loprinzi CL, Levin TR, Rex DK, Ahnen DJ, et al.  Stool DNA and occult blood testing for screen detection of colorectal neoplasia. Ann Intern Med. 2008; 149: 441–50, W81.
    
    [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.7326/0003-4819-149-7-200810070-00004&link_type=DOI) 
    
    [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=18838724&link_type=MED&atom=%2Fcbm%2F14%2F1%2F42.atom) 
    
    [Web of Science](http://www.cancerbiomed.org/lookup/external-ref?access_num=000260004400001&link_type=ISI) 

8.  8. Bonasio R, Tu S, Reinberg D.  Molecular signals of epigenetic states. Science. 2010; 330: 612–6.
    
    [Abstract/FREE Full Text](http://www.cancerbiomed.org/lookup/ijlink/YTozOntzOjQ6InBhdGgiO3M6MTQ6Ii9sb29rdXAvaWpsaW5rIjtzOjU6InF1ZXJ5IjthOjQ6e3M6ODoibGlua1R5cGUiO3M6NDoiQUJTVCI7czoxMToiam91cm5hbENvZGUiO3M6Mzoic2NpIjtzOjU6InJlc2lkIjtzOjEyOiIzMzAvNjAwNC82MTIiO3M6NDoiYXRvbSI7czoxNzoiL2NibS8xNC8xLzQyLmF0b20iO31zOjg6ImZyYWdtZW50IjtzOjA6IiI7fQ==) 

9.  9. Portela A, Esteller M.  Epigenetic modifications and human disease. Nat Biotechnol. 2010; 28: 1057–68.
    
    [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1038/nbt.1685&link_type=DOI) 
    
    [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=20944598&link_type=MED&atom=%2Fcbm%2F14%2F1%2F42.atom) 
    
    [Web of Science](http://www.cancerbiomed.org/lookup/external-ref?access_num=000282870500028&link_type=ISI) 

10. 10. Riddihough G, Zahn LM.  What is epigenetics? Science. 2010; 330: 611.
    
    [FREE Full Text](http://www.cancerbiomed.org/lookup/ijlink/YTozOntzOjQ6InBhdGgiO3M6MTQ6Ii9sb29rdXAvaWpsaW5rIjtzOjU6InF1ZXJ5IjthOjQ6e3M6ODoibGlua1R5cGUiO3M6NDoiRlVMTCI7czoxMToiam91cm5hbENvZGUiO3M6Mzoic2NpIjtzOjU6InJlc2lkIjtzOjEyOiIzMzAvNjAwNC82MTEiO3M6NDoiYXRvbSI7czoxNzoiL2NibS8xNC8xLzQyLmF0b20iO31zOjg6ImZyYWdtZW50IjtzOjA6IiI7fQ==) 

11. 11. Kulis M, Esteller M.  DNA methylation and cancer. Adv Genet. 2010; 70: 27–56.
    
    [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1016/B978-0-12-380866-0.60002-2&link_type=DOI) 
    
    [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=20920744&link_type=MED&atom=%2Fcbm%2F14%2F1%2F42.atom) 

12. 12. Rauch TA, Wu XW, Zhong XY, Riggs AD, Pfeifer GP.  A human B cell methylome at 100-base pair resolution. Proc Natl Acad Sci U S A. 2009; 106: 671–8.
    
    [Abstract/FREE Full Text](http://www.cancerbiomed.org/lookup/ijlink/YTozOntzOjQ6InBhdGgiO3M6MTQ6Ii9sb29rdXAvaWpsaW5rIjtzOjU6InF1ZXJ5IjthOjQ6e3M6ODoibGlua1R5cGUiO3M6NDoiQUJTVCI7czoxMToiam91cm5hbENvZGUiO3M6NDoicG5hcyI7czo1OiJyZXNpZCI7czo5OiIxMDYvMy82NzEiO3M6NDoiYXRvbSI7czoxNzoiL2NibS8xNC8xLzQyLmF0b20iO31zOjg6ImZyYWdtZW50IjtzOjA6IiI7fQ==) 

13. 13. Ashktorab H, Brim H.  DNA methylation and colorectal cancer. Curr Colorectal Cancer Rep. 2014; 10: 425–30.
    
    [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1007/s11888-014-0245-2&link_type=DOI) 

14. 14. Bariol C, Suter C, Cheong K, Ku SL, Meagher A, Hawkins N, et al.  The relationship between hypomethylation and cpg island methylation in colorectal neoplasia. Am J Pathol. 2003; 162: 1361–71.
    
    [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=12651628&link_type=MED&atom=%2Fcbm%2F14%2F1%2F42.atom) 
    
    [Web of Science](http://www.cancerbiomed.org/lookup/external-ref?access_num=000181748300034&link_type=ISI) 

15. 15. Toyota M, Ahuja N, Ohe-Toyota M, Herman JG, Baylin SB, Issa JPJ.  CpG island methylator phenotype in colorectal cancer. Proc Natl Acad Sci U S A. 1999; 96: 8681–6.
    
    [Abstract/FREE Full Text](http://www.cancerbiomed.org/lookup/ijlink/YTozOntzOjQ6InBhdGgiO3M6MTQ6Ii9sb29rdXAvaWpsaW5rIjtzOjU6InF1ZXJ5IjthOjQ6e3M6ODoibGlua1R5cGUiO3M6NDoiQUJTVCI7czoxMToiam91cm5hbENvZGUiO3M6NDoicG5hcyI7czo1OiJyZXNpZCI7czoxMDoiOTYvMTUvODY4MSI7czo0OiJhdG9tIjtzOjE3OiIvY2JtLzE0LzEvNDIuYXRvbSI7fXM6ODoiZnJhZ21lbnQiO3M6MDoiIjt9) 

16. 16. Migliore L, Migheli F, Spisni R, Coppedè F.  Genetics, cytogenetics, and epigenetics of colorectal cancer. J Biomed Biotechnol. 2011; 2011: 792362.
    
    [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1155/2011/792362&link_type=DOI) 
    
    [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=21490705&link_type=MED&atom=%2Fcbm%2F14%2F1%2F42.atom) 

17. 17. Issa JP.  CpG island methylator phenotype in cancer. Nat Rev Cancer. 2004; 4: 988–93.
    
    [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1038/nrc1507&link_type=DOI) 
    
    [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=15573120&link_type=MED&atom=%2Fcbm%2F14%2F1%2F42.atom) 
    
    [Web of Science](http://www.cancerbiomed.org/lookup/external-ref?access_num=000225454300017&link_type=ISI) 

18. 18. Terry MB, Neugut AI, Bostick RM, Sandler RS, Haile RW, Jacobson JS, et al.  Risk factors for advanced colorectal adenomas: A pooled analysis. Cancer Epidemiol Biomarkers Prev. 2002; 11: 622–9.
    
    [Abstract/FREE Full Text](http://www.cancerbiomed.org/lookup/ijlink/YTozOntzOjQ6InBhdGgiO3M6MTQ6Ii9sb29rdXAvaWpsaW5rIjtzOjU6InF1ZXJ5IjthOjQ6e3M6ODoibGlua1R5cGUiO3M6NDoiQUJTVCI7czoxMToiam91cm5hbENvZGUiO3M6NDoiY2VicCI7czo1OiJyZXNpZCI7czo4OiIxMS83LzYyMiI7czo0OiJhdG9tIjtzOjE3OiIvY2JtLzE0LzEvNDIuYXRvbSI7fXM6ODoiZnJhZ21lbnQiO3M6MDoiIjt9) 

19. 19. Galamb O, Kalmar A, Péterfia B, Csabai I, Bodor A, Ribli D, et al.  Aberrant DNA methylation of wnt pathway genes in the development and progression of cimp-negative colorectal cancer. Epigenetics. 2016; 11: 588–602.
    
    [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1080/15592294.2016.1190894&link_type=DOI) 

20. 20. Esteve P, Bovolenta P.  The advantages and disadvantages of SFRP1 and SFRP2 expression in pathological events. Tohoku J Exp Med. 2010; 221: 11–7.
    
    [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1620/tjem.221.11&link_type=DOI) 
    
    [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=20448436&link_type=MED&atom=%2Fcbm%2F14%2F1%2F42.atom) 

21. 21. Suzuki H, Gabrielson E, Chen W, Anbazhagan R, van Engeland M, Weijenberg MP, et al.  A genomic screen for genes upregulated by demethylation and histone deacetylase inhibition in human colorectal cancer. Nat Genet. 2002; 31: 141–9.
    
    [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1038/ng892&link_type=DOI) 
    
    [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=11992124&link_type=MED&atom=%2Fcbm%2F14%2F1%2F42.atom) 
    
    [Web of Science](http://www.cancerbiomed.org/lookup/external-ref?access_num=000175903500009&link_type=ISI) 

22. 22. Suzuki H, Watkins DN, Jair KW, Schuebel KE, Markowitz SD, Chen WD, et al.  Epigenetic inactivation of *SFRP* genes allows constitutive WNT signaling in colorectal cancer. Nat Genet. 2004; 36: 417–22.
    
    [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1038/ng1330&link_type=DOI) 
    
    [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=15034581&link_type=MED&atom=%2Fcbm%2F14%2F1%2F42.atom) 
    
    [Web of Science](http://www.cancerbiomed.org/lookup/external-ref?access_num=000220647200028&link_type=ISI) 

23. 23. Caldwell GM, Jones C, Gensberg K, Jan S, Hardy RG, Byrd P, et al.  The WNT antagonist *sFRPl* in colorectal tumorigenesis. Cancer Res. 2004; 64: 883–8.
    
    [Abstract/FREE Full Text](http://www.cancerbiomed.org/lookup/ijlink/YTozOntzOjQ6InBhdGgiO3M6MTQ6Ii9sb29rdXAvaWpsaW5rIjtzOjU6InF1ZXJ5IjthOjQ6e3M6ODoibGlua1R5cGUiO3M6NDoiQUJTVCI7czoxMToiam91cm5hbENvZGUiO3M6NjoiY2FucmVzIjtzOjU6InJlc2lkIjtzOjg6IjY0LzMvODgzIjtzOjQ6ImF0b20iO3M6MTc6Ii9jYm0vMTQvMS80Mi5hdG9tIjt9czo4OiJmcmFnbWVudCI7czowOiIiO30=) 

24. 24. Chen YZ, Liu D, Zhao YX, Wang HT, Gao Y, Chen Y.  Aberrant promoter methylation of the *SFRPl* gene may contribute to colorectal carcinogenesis: A meta-analysis. Tumor Biol. 2014; 35: 9201–10.
    
    

25. 25. Kalmár A, Péterfia B, Hollósi P, Galamb O, Spisák S, Wichmann B, et al.  DNA hypermethylation and decreased mRNA expression of *MAL, PRIMAI, PTGDR* and *SFRPl* in colorectal adenoma and cancer. BMC Cancer. 2015; 15: 736.
    
    

26. 26. Nagasaka T, Tanaka N, Cullings HM, Sun DS, Sasamoto H, Uchida T, et al.  Analysis of fecal DNA methylation to detect gastrointestinal neoplasia. J Natl Cancer Inst. 2009; 101: 1244–58.
    
    [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1093/jnci/djp265&link_type=DOI) 
    
    [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=19700653&link_type=MED&atom=%2Fcbm%2F14%2F1%2F42.atom) 
    
    [Web of Science](http://www.cancerbiomed.org/lookup/external-ref?access_num=000270015400007&link_type=ISI) 

27. 27. Wang DR, Tang D.  Hypermethylated *SFRP2* gene in fecal DNA is a high potential biomarker for colorectal cancer noninvasive screening. World J Gastroenterol. 2008; 14: 524–31.
    
    [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.3748/wjg.14.524&link_type=DOI) 
    
    [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=18203283&link_type=MED&atom=%2Fcbm%2F14%2F1%2F42.atom) 

28. 28. Huang ZH, Li LH, Wang JF.  Hypermethylation of *SFRP2* as a potential marker for stool-based detection of colorectal cancer and precancerous lesions. Dig Dis Sci. 2007; 52: 2287–91.
    
    [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1007/s10620-007-9755-y&link_type=DOI) 
    
    [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=17410438&link_type=MED&atom=%2Fcbm%2F14%2F1%2F42.atom) 
    
    [Web of Science](http://www.cancerbiomed.org/lookup/external-ref?access_num=000248810300044&link_type=ISI) 

29. 29. Leung WK, To KF, Man EPS, Chan MWY, Hui AJ, Ng SS, et al.  Detection of hypermethylated DNA or cyclooxygenase-2 messenger RNA in fecal samples of patients with colorectal cancer or polyps. Am J Gastroenterol. 2007; 102: 1070–6.
    
    [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1111/j.1572-0241.2007.01108.x&link_type=DOI) 
    
    [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=17378912&link_type=MED&atom=%2Fcbm%2F14%2F1%2F42.atom) 

30. 30. Huang ZH, Li LH, Yang F, Wang JF.  Detection of aberrant methylation in fecal DNA as a molecular screening tool for colorectal cancer and precancerous lesions. World J Gastroenterol. 2007; 13: 950–4.
    
    [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=17352030&link_type=MED&atom=%2Fcbm%2F14%2F1%2F42.atom) 
    
    [Web of Science](http://www.cancerbiomed.org/lookup/external-ref?access_num=000244879200023&link_type=ISI) 

31. 31. Müller HM, Oberwalder M, Fiegl H, Morandell M, Goebel G, Zitt M, et al.  Methylation changes in faecal DNA: A marker for colorectal cancer screening? Lancet. 2004; 363: 1283–5.
    
    [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1016/S0140-6736(04)16002-9&link_type=DOI) 
    
    [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=15094274&link_type=MED&atom=%2Fcbm%2F14%2F1%2F42.atom) 
    
    [Web of Science](http://www.cancerbiomed.org/lookup/external-ref?access_num=000220904900011&link_type=ISI) 

32. 32. Tang D, Liu J, Wang DR, Yu HF, Li YK, Zhang JQ.  Diagnostic and prognostic value of the methylation status of secreted frizzled- related protein 2 in colorectal cancer. Clin Invest Med. 2011; 34: E88–95.
    
    [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=21463549&link_type=MED&atom=%2Fcbm%2F14%2F1%2F42.atom) 

33. 33. Chang E, Park DI, Kim YJ, Kim BK, Park JH, Kim HJ, et al.  Detection of colorectal neoplasm using promoter methylation of ITGA4, SFRP2, and p16 in stool samples: A preliminary report in Korean patients. Hepatogastroenterology. 2010; 57: 720–7.
    
    [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=21033217&link_type=MED&atom=%2Fcbm%2F14%2F1%2F42.atom) 

34. 34. Kim MS, Louwagie J, Carvalho B, Terhaar Sive Droste JS, Park HL, Chae YK, et al.  Promoter DNA methylation of oncostatin m receptor-beta as a novel diagnostic and therapeutic marker in colon cancer. PLoS One. 2009; 4: e6555.
    
    

35. 35. Zhang W, Bauer M, Croner RS, Pelz JO, Lodygin D, Hermeking H, et al.  DNA stool test for colorectal cancer: Hypermethylation of the secreted frizzled-related protein-1 gene. Dis Colon Rectum. 2007; 50: 1618–26; discussion 1626-7.
    
    [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1007/s10350-007-0286-6&link_type=DOI) 
    
    [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=17762966&link_type=MED&atom=%2Fcbm%2F14%2F1%2F42.atom) 

36. 36. Sui C, Ma J, Chen Q, Yang Y.  The variation trends of SFRP2 methylation of tissue, feces, and blood detection in colorectal cancer development. Eur J Cancer Prev. 2015; 25: 288–98.
    
    

37. 37. Zhang X, Song YF, Lu HN, Wang DP, Zhang XS, Huang SL, et al.   Combined detection of plasma gata5 and sfrp2 methylation is a valid noninvasive biomarker for colorectal cancer and adenomas. World J Gastroenterol. 2015; 21: 2629–37.
    
    

38. 38. Lu HN, Huang SL, Zhang X, Wang DP, Zhang XS, Yuan XG, et al.  DNA methylation analysis of SFRP2, GATA4/5, NDRG4 and vim for the detection of colorectal cancer in fecal DNA. Oncol Lett. 2014; 8: 1751–6.
    
    

39. 39. Sui CG, Wang G, Chen Q, Ma JZ.  Variation risks of SFRP2 hypermethylation between precancerous disease and colorectal cancer. Tumor Biol. 2014; 35: 10457–65.
    
    

40. 40. Salehi R, Mohammadi M, Emami MH, Salehi AR.  Methylation pattern of SFRP1 promoter in stool sample is a potential marker for early detection of colorectal cancer. Adv Biomed Res. 2012; 1: 87.
    
    

41. 41. Yang QH, Huang T, Ye GL, Wang BJ, Zhang XJ.  Methylation of SFRP2 gene as a promising noninvasive biomarker using feces in colorectal cancer diagnosis: A systematic meta-analysis. Sci Rep. 2016; 6: 33339.
    
    

42. 42. Zhang H, Qi J, Wu YQ, Zhang P, Jiang J, Wang QX, et al.  Accuracy of early detection of colorectal tumours by stool methylation markers: A meta-analysis. World J Gastroenterol. 2014; 20: 14040–50.
    
    [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=25320544&link_type=MED&atom=%2Fcbm%2F14%2F1%2F42.atom) 

43. 43. Sierko E, Wojtukiewicz MZ, Kisiel W.  The role of tissue factor pathway inhibitor-2 in cancer biology. Semin Thromb Hemost. 2007; 33: 653–9.
    
    [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1055/s-2007-991532&link_type=DOI) 
    
    [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=18000791&link_type=MED&atom=%2Fcbm%2F14%2F1%2F42.atom) 
    
    [Web of Science](http://www.cancerbiomed.org/lookup/external-ref?access_num=000251062900004&link_type=ISI) 

44. 44. Glöckner SC, Dhir M, Yi JM, McGarvey KE, Van Neste L, Louwagie J, et al.  Methylation of *TFPI2* in stool DNA: A potential novel biomarker for the detection of colorectal cancer. Cancer Res. 2009; 69: 4691–9.
    
    [Abstract/FREE Full Text](http://www.cancerbiomed.org/lookup/ijlink/YTozOntzOjQ6InBhdGgiO3M6MTQ6Ii9sb29rdXAvaWpsaW5rIjtzOjU6InF1ZXJ5IjthOjQ6e3M6ODoibGlua1R5cGUiO3M6NDoiQUJTVCI7czoxMToiam91cm5hbENvZGUiO3M6NjoiY2FucmVzIjtzOjU6InJlc2lkIjtzOjEwOiI2OS8xMS80NjkxIjtzOjQ6ImF0b20iO3M6MTc6Ii9jYm0vMTQvMS80Mi5hdG9tIjt9czo4OiJmcmFnbWVudCI7czowOiIiO30=) 

45. 45. Zhang JP, Wang J, Gui YL, Zhu QQ, Xu ZW, Li JS.  human stool vimentin, oncostatin M receptor and tissue factor pathway inhibitor 2 gene methylation analysis for the detection of colorectal neoplasms. Natl Med J China. 2011; 91: 2482–4.
    
    

46. 46. Zhang JP, Yang SB, Xie YY, Chen XY, Zhao Y, He DZ, et al.  Detection of methylated tissue factor pathway inhibitor 2 and human long DNA in fecal samples of patients with colorectal cancer in China. Cancer Epidemiol. 2012; 36: 73–7.
    
    [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=21621497&link_type=MED&atom=%2Fcbm%2F14%2F1%2F42.atom) 

47. 47. Li YW, Kong FM, Zhou JP, Dong M.  Aberrant promoter methylation of the vimentin gene may contribute to colorectal carcinogenesis: A meta-analysis. Tumor Biol. 2014; 35: 6783–90.
    
    

48. 48. Li M, Chen WD, Papadopoulos N, Goodman SN, Bjerregaard NC, Laurberg S, et al.  Sensitive digital quantification of DNA methylation in clinical samples. Nat Biotechnol. 2009; 27: 858–63.
    
    [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1038/nbt.1559&link_type=DOI) 
    
    [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=19684580&link_type=MED&atom=%2Fcbm%2F14%2F1%2F42.atom) 
    
    [Web of Science](http://www.cancerbiomed.org/lookup/external-ref?access_num=000269751400029&link_type=ISI) 

49. 49. Baek YH, Chang E, Kim YJ, Kim BK, Sohn JH, Park DI.  Stool methylation-specific polymerase chain reaction assay for the detection of colorectal neoplasia in korean patients. Dis Colon Rectum. 2009; 52: 1452–9; discussion 1459–63.
    
    [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1007/DCR.0b013e3181a79533&link_type=DOI) 
    
    [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=19617759&link_type=MED&atom=%2Fcbm%2F14%2F1%2F42.atom) 
    
    [Web of Science](http://www.cancerbiomed.org/lookup/external-ref?access_num=000273644600016&link_type=ISI) 

50. 50. Shirahata A, Sakata M, Sakuraba K, Goto T, Mizukami H, Saito M, et al.  Vimentin methylation as a marker for advanced colorectal carcinoma. Anticancer Res. 2009; 29: 279–81.
    
    [Abstract/FREE Full Text](http://www.cancerbiomed.org/lookup/ijlink/YTozOntzOjQ6InBhdGgiO3M6MTQ6Ii9sb29rdXAvaWpsaW5rIjtzOjU6InF1ZXJ5IjthOjQ6e3M6ODoibGlua1R5cGUiO3M6NDoiQUJTVCI7czoxMToiam91cm5hbENvZGUiO3M6MTA6ImFudGljYW5yZXMiO3M6NToicmVzaWQiO3M6ODoiMjkvMS8yNzkiO3M6NDoiYXRvbSI7czoxNzoiL2NibS8xNC8xLzQyLmF0b20iO31zOjg6ImZyYWdtZW50IjtzOjA6IiI7fQ==) 

51. 51. Chen WD, Han ZJ, Skoletsky J, Olson J, Sah J, Myeroff L, et al.  Detection in fecal DNA of colon cancer-specific methylation of the nonexpressed vimentin gene. J Natl Cancer Inst. 2005; 97: 1124–32.
    
    [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1093/jnci/dji204&link_type=DOI) 
    
    [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=16077070&link_type=MED&atom=%2Fcbm%2F14%2F1%2F42.atom) 
    
    [Web of Science](http://www.cancerbiomed.org/lookup/external-ref?access_num=000232272700008&link_type=ISI) 

52. 52. Xiao ZJ, Li BS, Wang GZ, Zhu WS, Wang ZQ, Lin JF, et al.  Validation of methylation-sensitive high-resolution melting (MS- HRM) for the detection of stool DNA methylation in colorectal neoplasms. Clin Chim Acta. 2014; 431: 154–63.
    
    

53. 53. Christmann M, Verbeek B, Roos WP, Kaina B.  O6-methylguanine- DNA methyltransferase (MGMT) in normal tissues and tumors: Enzyme activity, promoter methylation and immunohistochemistry. Biochim Biophys Acta. 2011; 1816: 179–90.
    
    [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=21745538&link_type=MED&atom=%2Fcbm%2F14%2F1%2F42.atom) 

54. 54. Zheng CG, Jin C, Ye LC, Chen NZ, Chen ZJ.  Clinicopathological significance and potential drug target of O6-methylguanine-DNA methyltransferase in colorectal cancer: A meta-analysis. Tumor Biol. 2015; 36: 5839–48.
    
    

55. 55. Li Y, Lyu Z, Zhao L, Cheng H, Zhu D, Gao Y, et al.  Prognostic value of MGMT methylation in colorectal cancer: A meta-analysis and literature review. Tumor Biol. 2015; 36: 1595–601.
    
    

56. 56. Inno A, Fanetti G, Di Bartolomeo M, Gori S, Maggi C, Cirillo M, et al.  Role of MGMT as biomarker in colorectal cancer. World J Clin Cases. 2014; 2: 835–9.
    
    [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.12998/wjcc.v2.i12.835&link_type=DOI) 
    
    [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=25516857&link_type=MED&atom=%2Fcbm%2F14%2F1%2F42.atom) 

57. 57. Kang YP, Cao FA, Chang WJ, Lou Z, Wang H, Wu LL, et al.  Gene methylation in stool for the screening of colorectal cancer and pre- malignant lesions. Chin J Gastroint Surg. 2011; 14: 52–6.
    
    

58. 58. Traverso G, Shuber A, Levin B, Johnson C, Olsson L, Schoetz DJ Jr., et al.  Detection of *APC* mutations in fecal DNA from patients with colorectal tumors. N Engl J Med. 2002; 346: 311–20.
    
    [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1056/NEJMoa012294&link_type=DOI) 
    
    [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=11821507&link_type=MED&atom=%2Fcbm%2F14%2F1%2F42.atom) 
    
    [Web of Science](http://www.cancerbiomed.org/lookup/external-ref?access_num=000173545300003&link_type=ISI) 

59. 59. Ahlquist DA, Skoletsky JE, Boynton KA, Harrington JJ, Mahoney DW, Pierceall WE, et al.  Colorectal cancer screening by detection of altered human DNA in stool: Feasibility of a multitarget assay panel. Gastroenterology. 2000; 119: 1219–27.
    
    [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1053/gast.2000.19580&link_type=DOI) 
    
    [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=11054379&link_type=MED&atom=%2Fcbm%2F14%2F1%2F42.atom) 
    
    [Web of Science](http://www.cancerbiomed.org/lookup/external-ref?access_num=000090060900009&link_type=ISI) 

60. 60. Feinberg AP, Vogelstein B.  Hypomethylation distinguishes genes of some human cancers from their normal counterparts. Nature. 1983; 301: 89–92.
    
    [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1038/301089a0&link_type=DOI) 
    
    [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=6185846&link_type=MED&atom=%2Fcbm%2F14%2F1%2F42.atom) 
    
    [Web of Science](http://www.cancerbiomed.org/lookup/external-ref?access_num=A1983PW91500052&link_type=ISI) 

61. 61. Esteller M.  Epigenetics in cancer. N Engl J Med. 2008; 358: 1148–59.
    
    [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1056/NEJMra072067&link_type=DOI) 
    
    [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=18337604&link_type=MED&atom=%2Fcbm%2F14%2F1%2F42.atom) 
    
    [Web of Science](http://www.cancerbiomed.org/lookup/external-ref?access_num=000253877200008&link_type=ISI) 

62. 62. Ide T, Kitajima Y, Ohtaka K, Mitsuno M, Nakafusa Y, Miyazaki K.  Expression of the hMLH1 gene is a possible predictor for the clinical response to 5-fluorouracil after a surgical resection in colorectal cancer. Oncol Rep. 2008; 19: 1571–6.
    
    [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=18497967&link_type=MED&atom=%2Fcbm%2F14%2F1%2F42.atom) 
    
    [Web of Science](http://www.cancerbiomed.org/lookup/external-ref?access_num=000256281300030&link_type=ISI) 

63. 63. Gaj T, Gersbach CA, Barbas CF.  ZFN, TALEN, and CRISPR/Cas- based methods for genome engineering. Trends Biotechnol. 2013; 31: 397–405.
    
    [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1016/j.tibtech.2013.04.004&link_type=DOI) 
    
    [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=23664777&link_type=MED&atom=%2Fcbm%2F14%2F1%2F42.atom) 
    
    [Web of Science](http://www.cancerbiomed.org/lookup/external-ref?access_num=000321175200004&link_type=ISI) 

64. 64. Fujita T, Fujii H.  Applications of engineered DNA-binding molecules such as TAL proteins and the CRISPR/Cas system in biology research. Int J Mol Sci. 2015; 16: 23143–64.
    
    

65. 65. Fujita T, Asano Y, Ohtsuka J, Takada Y, Saito K, Ohki R, et al.  Identification of telomere-associated molecules by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP). Sci Rep. 2013; 3: 3171.
    
    [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1038/srep03171&link_type=DOI) 
    
    [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=24201379&link_type=MED&atom=%2Fcbm%2F14%2F1%2F42.atom) 

66. 66. Fujita T, Fujii H.  Efficient isolation of specific genomic regions and identification of associated proteins by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using crispr. Biochem Biophys Res Commun. 2013; 439: 132–6.
    
    [CrossRef](http://www.cancerbiomed.org/lookup/external-ref?access_num=10.1016/j.bbrc.2013.08.013&link_type=DOI) 
    
    [PubMed](http://www.cancerbiomed.org/lookup/external-ref?access_num=23942116&link_type=MED&atom=%2Fcbm%2F14%2F1%2F42.atom)